nghiên cứu nâng cao khả năng sinh tổng hợp kháng sinh từ streptomyces 173.113

7/23/2019 Nghin Cu Nng Cao Kh Nng Sinh Tng Hp Khng Sinh T Streptomyces 173.113

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B GIO DC V O TO B Y T

TRNG I HC DC H NI

PHAN TH HUYN

NGHIN CU NNG CAO KHNNG SINH TNG HP KHNGSINH TSTREPTOMYCES 173.113

LUN VN THC S DC HC

H NI 2014


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B GIO DC V O TO B Y T

TRNG I HC DC H NI

PHAN TH HUYN

NGHIN CU NNG CAO KH NNGSINH TNG HP KHNG SINH T

STREPTOMYCES 173.113

LUN VN THC S DC HC

CHUYN NGNH: CNG NGH DC PHM BO CHTHUC

M S: 60720402

Ngi hng dn khoa hc: PGS.TS Cao Vn Thu

H NI 2014


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Li cm n

Ti xin gi li cm n su sc nht n thy gio PGS.TSCao Vn

Thungi tn tnh hng dn ti t nhng bc u tin cho n khi ti

hon thnh lun vn ny.

Ti xin chn thnh cm n ti thy c gio, cc cn b k thut vin

ang ging dy v lm vic ti b mn Vi sinh - Sinh hc trng i hc

Dc H ni tn tnh gip ti trong sut thi gian ti lm lun vn ti

b mn.

Nhn dp ny ti cng xin gi li cm n ti Ban gim hiu cng ton

th cc thy c gio trng i hc Dc H Ni dy d v to iu kinthun li cho ti trong sut qu trnh hc tp v rn luyn ti trng.

V cui cng l li cm n ti gi ti gia nh, bn b ng vin,

gip ti trong sut thi gian thc hin lun vn.

Do thi gian thc nghim hn hp, iu kin trang thit b, phng tin

nghin cu cng nh trnh ca bn thn cn hn ch, lun vn ny khng

trnh khi cn nhiu thiu st. Ti rt mong nhn c s gp ca thy c

v bn b lun vn ny c th c hon thin hn.

Ti xin chn thnh cm n!

H Ni, thng 10 nm 2014

Hc vin

Phan Th Huyn


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MC LC

T VN

CHNG 1. TNG QUAN1.1. i cng v Streptomyces

1.1.1. c im ca x khun chi Streptomyces

1.1.2. Kh nng sinh tng hp khng sinh ca Streptomyces

1.1.3. S tng qut sinh tng hp khng sinh t x khun..

1.1.4. Nhng im lu khi nghin cu cc cht khng sinh t x khun

1.2. Bc u nghin cu ln men sinh tng hp khng sinh nh Streptomyces 173.113

1.3. Nghin cu tng hp Actinomycin X2 t Streptomyces 15.29 Streptomycesmicroflavus..

1.4. Cng ngh ln men Daunorubicin v Adriamycin.

1.5. Tng hp cirramycin B tStreptomyces cyaneus.

1.6. Khng sinh chng nm non-polyen tStreptomyces albidoflavus.

1.7. Tng hp khng sinh antimycin A9 tStreptomyces Sp. K01-0031

1.8. Sparsomycin khng sinh c tng hp tStreptomyces Sp. AZ-NIOFD1.

CHNG2:

I TNG V PHNG PHP NGHIN CU.2.1. Nguyn vt liu v thit b

2.1.1. Nguyn vt liu

2.1.2. My mc thit b.

2.2. Phng php thc nghim

2.2.1. Nui cy v gi ging x khun.

2.2.2. nh gi hot tnh khng sinh bng phng php khuch tn...

2.2.3. Sng lc ngu nhin

2.2.4. t bin bng nh sng UV

2.2.5. Ln men chm tng hp khng sinh ..

2.2.6. Chit khng sinh t dch ln men bng dung mi hu c ..

2.2.7. Tch cc thnh phn trong khng sinh bng sc k lp mng.

2.2.8. Thu khng sinh th bng phng php ct quay chn khng

1

22

2

3

4

5

5

6

7

9

9

1

12

1414

14

1

1

1

1

1

1

2

2

2

22
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2.2.9. Tinh ch khng sinh th bng sc k ct

2.2.10. Xc nh cu trc ha hc thnh khng sinh chnh tinh khit thu c

CHNG 3: KT QU THC NGHIM V NHN XT

3.1. Chn lc v ci to ging x khun3.1.1. Kt qu sng lc ngu nhin ..

3.1.2. Kt qu t bin ln th nht ..

3.1.3. Kt qu t bin ln th hai ..

3.2. Xc nh iu kin ln men, chit tch khng sinh tt nht...

3.2.1. Kt qu chn chng ln men ..

3.2.2. Kho st chn mi trng ln men chm.

3.2.3. Kt qu ln men, chit v ct quay thu khng sinh th.3.2.4. Kt qu tinh ch khng sinh ln th nht

3.2.5. Kho st h dung mi tinh ch khng sinh ln th hai

3.2.6. Kt qu tinh ch khng sinh ln th hai.

3.2.7. Kho st h dung mi tinh ch khng sinh ln th ba.

3.2.8. Kt qu tinh ch khng sinh ln th ba..

3.3. Nghin cu tnh cht, cu trc khng sinh.

3.3.1. Kt qu o nng chy.

3.3.2. Kt qu ph t ngoi .

3.3.3. Kt qu ph hng ngoi ..

3.3.4. Kt qu o ph khi .

3.3.5. Kt qu o ph cng hngt ht nhn

CHNG 4. BN LUN

4.1. Chn lc v ci to ging x khun

4.2. Xc nh iu kin ln men, chit tch khng sinh ti thch .

4.3. Nghin cu cu trc khng sinh .

KT LUN V KIN NGH..

TI LIU THAM KHO

PH LC

22

2

24

2424

2

2

2

2

3

3232

3

3

3

4

4

4

4

44

4

4

4

4

5

52

54
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DANH MC CC BNG

Bng 1.1: Gi tr MIC v MFC ca khng sinh t Streptomyces. albidoflavus.......

Bng 1.2: Gi tr MIC ca cc antimycin trn Caenorhabditis elegans v

Artemia salina ............................ ........................... ........................... ..................

Bng 2.1: Cc vi sinh vt kim nh .......................... ............................ ..............

Bng 2.2.: Cc MT nui cy VSV kim nh...........

Bng 2.3.: Cc dung mi s dng

Bng 3.1: Kt qu th HTKS sng lc ngu nhin ........................ .......................

Bng 3.2.: Tnh ton t l sng st sau t bin ln th nht

Bng 3.3: Kt qu th HTKS t bin ln 1 .............. ............................ ..............

Bng 3.4.: Tnh ton t l sng st sau t bin ln th hai

Bng 3.5: Kt qu th HTKS t bin ln 2 .............. ............................ ..............

Bng 3.6: Kt qu chn chng ln men .......................... ........................... .........

Bng 3.7.: Kt qu chn mi trng ln men...........

Bng 3.8: Kt qu sc k ct ln th nht .......................................... ..

Bng 3.9: Kt qu SKLM cc phn on chy ct ln 1 .....................................

Bng 3.10: Kt qu SKLM cc phn on kho st dung mi tinh ch KS ln 2 Bng 3.11: T l cc thnh phn h dung mi Ethylacetat: methanol: n-hexan .......

Bng 3.12: Kt qu SKLM vi cc h dung mi Ethylacetat: methanol: n-hexan

Bng 3.13: Kt qu th hot tnh KS ca cc phn on sau tinh ch ln 2 ..........

Bng 3.14: Kt qu SKLM cc phn on sau tinh ch ln th hai ......................

Bng 3.15: Thnh phn cc h dung mi tinh ch khng sinh ln 3 ....................

Bng 3.16.: Kt qu SKLM vi cc h dung mi tinh ch khng sinh ln th 3.

Bng 3.17.: Kt qu th HTKS cc phn on sau tinh ch ln 3

Bng 3.18.: Kt qu SKLM cc phn on sau tinh ch ln th 3

Bng 3.19.: Bin gii cc nhm chc ca KS1 t ph IR

Bng 3.20.: Bin gii cc nhm chc ca KS2 t ph IR

Bng 3.21.: So snh ph NMR ca KS2 vi khung phenoxazone ca actinomycin D

10

11

14

15

15

24

25

26

28

28

30

31

33

34

3636

37

38

38

40

40

41

42

44

45

48


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DANH MC CC HNH

Hnh 1.1 : Mt s khng sinh c tng hp t Streptomyces............... 3

Hnh 1.2 : S ln men tng hp khng sinh ...................................... 4

Hnh P1 : Ph UV ca cht khng sinh KS1

Hnh P2 : Ph UV ca cht khng sinh KS2

Hnh P3 : Ph IR khng sinh KS1

Hnh P4 : Ph IR khng sinh KS2

Hnh P5 : Ph khi MS khng sinh KS1

Hnh P6 : Ph khi MS khng sinh KS2

Hnh P7 : Ph 1H NMR ca khng sinh KS2

Hnh P7a, b, c: Ph 1H NMR gin ca khng sinh KS2

Hnh P8 : Ph 13C NMR ca khng sinh KS2

Hnh P8a, b, c : Ph 13C NMR gin ca khng sinh KS2

Hnh P9 : Ph COSY ca khng sinh KS2

Hnh P9a, b, c : Ph COSY gin ca khng sinh KS2Hnh P10 : Ph DEPT ca khng sinh KS2

Hnh P10a, b : Ph DEPT gin ca khng sinh KS2

Hnh P11 : Ph HSQC ca khng sinh KS2

Hnh P11a, b : Ph HSQC gin ca khng sinh KS2

Hnh P12 : Ph HMBC ca khng sinh KS2

Hnh P12a, b, c, d : Ph HMBC gin ca khng sinh KS2


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1

T VN

Vi tnh cht l mt nc c kh hu nhit i m gi ma, Vit Nam nm trong

s cc nc c nhu cu s dng khng sinh rt cao.Tuy nhin, trn th trng dcphm nc ta hin nay, hu ht cc thuc khng sinh u c nhp khu dng thnh

phm hoc bn thnh phm, ngnh cng nghip sn xut khng sinh cha thc s hnh

thnh. V vy, n Quy hoch chi tit pht trin cng nghip Dc Vit Nam giai

on n nm 2020 v tm nhn n nm 2030 xc nh y mnh vic sn xut

nguyn liu khng sinh th h mi p ng nhu cu v nguyn liu sn xut khng

sinh trong nc[4].

Tng hp ho hc, bn tng hp v sinh tng hp l ba hng pht hin v

tng hp khng sinh. Trong sinh tng hp khng sinh vi nhiu u im c s

dng ch yu tm ra khng sinh mi.

Trong tt c khng sinh c bit n hin nay, c ti 55% khng sinh c

phn lp t x khun, 75% trong s t chi Streptomyces [8, 23]. B mn Vi sinh

Sinh hc trng i hc Dc H Ni u t nghin cu vpht hin nhiu chng

x khun phn lp t t, bn, c kh nng sinh tng hp khng sinh, trong c

chng x khun Streptomyces 173.113. Bc u nghin cu cho thy Streptomyces

173.113 c kh nng sinh tng hp khng sinh ph rng, tuy nhin hiu sut tng hp

cng nh hiu sut tinh ch cha cao, cha bin giic cu trc ha hc cakhng

sinh thu c[7]. Bi vy, chng ti la chn ti Nghin cu nng cao kh nng

sinh tng hp khng sinh tStreptomyces 173.113lm lun vn thc s vicc mc

tiu c th nh sau:

1.

Chn lc, ci to ging nng cao hiu sut ln men sinh tng hp khng

sinh.

2. Nng cao kh nngln men, tch chit, tinh ch khng sinh.

3.

Bc u nghin cu cutrc khng sinh chnh thu c.


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CHNG 1. TNG QUAN

1.1. i cng vStreptomyces

1.1.1. c im ca x khun chi StreptomycesCc loi x khun thuc Streptomycesc kh nng to ra nhiu khng sinh

c cu trc phc tp. Trong tng s cc khng sinh c tm thy do x khun

tng hp th c ti 75% l t Streptomyces[8, 23].

- c im hnh thi:h si ca x khun gm c khun ty c cht v

khun ty kh sinh:

+ Khun ty c cht: mc su vo mi trng nui cy, khng phn ct

trong sut qu trnh pht trin, b mt nhn hoc sn si, c th tit ra m i

trng mt s loi sc t, c sc t tan trong nc, c sc t ch tan trong dung

mi hu c.

+ Khun ty kh sinh: do khun ty c cht pht trin di ra trong khng kh.

Sau mt thi gian pht trin trn nh khun ty kh sinh s xut hin cc chui

bo t.

+ Chui bo t (si bo t) c rt nhiu hnh dng khc nhau: thng, sng,

mc cu, xon Chui bo t phn chia to thnh cc bo t trn, l c quan

sinh sn ch yu ca x khun. B mt bo t c th c dng trn nhn (sm), x

x da cc (wa), c gai (sp) hoc c tc (sp).

-c im sinh l:Streptomycesl sinh vt d dng, c tnh oxi ha cao.

pht trin, chng phn gii cc hydratcarbon lm ngun cung cp vt cht v

nng lng, ng thi thy phn cc hp cht nh gelatin, casein, kh nitratthnh nitrit. Streptomycesl loi x khun h hp hiu kh. Nhit ti thch ca

chng l 25-30C, pH ti thch thng l 6,8-7,5.


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-Kh nng to sc t:Sc t to thnh t Streptomycesc chia lm 4

loi: sc t ha tan, sc t ca khun ty c cht, sc t ca khun ty kh sinh, sc

t melanoid[1, 3, 5, 27].

1.1.2. Kh nng sinh tng hp khng sinh ca Streptomyces

Trong s cc khng sinh do x khun tng hp c bit n hin nay th

phn ln c tng hp t Streptomyces. Cc cht khng sinh c tng hp t

x khun bao gm cc cht chng nm, cc cht khng sinh, v ha cht tr liu

ha hc trong ung th. Hnh 1 di y gii thiu mt s khng sinh, trong

phn ln ldo Streptomycestng hp[2, 8, 23].

Hnh 1.1: Mt s khng sinh c tng hp t Streptomyces


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1.1.3. S tng qut sinh tng hp khng sinh t x khun

Cc bc c bn trong qu trnh ln men sn xut khng sinh t x khun c

th hin trong hnh 1.2 di y.

Hnh 1.2: S tng qut ln men sn xut khng sinh

Ging truyn trong phng th nghim

Bnh nhn ging trong phng th nghim

Bnh nhn ging trn thit b nhn ging

Bnh ln men to khng sinh

Dch ln men

Dch lcSinh khi

Sinh khi th

Dch chit sinh khi

Sn phm tinh ch

Dch chit khng sinh

Dch chit m c

Sn phm tinh ch

Sn phm kim nghim

Sn phm c ng gi


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1.1.4. Nhng im lu khi nghin cu cc cht khng sinh t x khun

X khun l vi khun tht thucnhm c th nhn s, nhng pht trin

thnh h si v thng sinh sn bng bo t. Ging nh nm si, x khun c

kh nng pht trin tt trn mi trng xp. Hin nay, do iu kin k thut m

bo mi trng nui v trng, ngi ta rt quan tm n vic nghin cu pht

trin phng php ln men trn mi trng xp sinh tng hp enzyme, cc

cht khng sinh v cc cht c hot tnh sinh hc khc.

X khun c cu to ging vi khun, nhng thi gian sinh trng pht

trin chm hn vi khun. Do , thi gian sinh trng, pht trin v sinh tng

hp cc cht khng sinh thng ko di n 96 -120 gi ln men.

Hu ht cc chng x khun pht trin tt trong mi trng trung tnh v

nhit ti u l 28-300C. Hn na, sau giai on sinh trng, pht trin, x

khun thng chuyn sang giai on t phn, do trong qu trnh sn xut phi

lu n thi gian thu nhn sn phm.[8]

1.2. Bc u nghin cu ln men sinh tng hp khng sinh t

Streptomyces 173.113

Streptomyces 173.113c b mn Vi sinh Sinh hc trng H Dc

H Ni phn lp t t Ty Nguyn. y l mt chng x khun n nh, c kh

nng tng hp khng sinh ph rng trn c vi khun Gram dng v Gram m,

c tim nng trong vic ng dng trong lnh vc phng v cha bnh. Tuy nhin,

hot tnh khng sinh cha cao, kh nng sinh tng hp khng sinh cn thp, do

cn phi tin hnh ci to ging la chn chng c kh nng sinh tng hp

khng sinh cao v nghin cu phng php tinh ch khng sinh thu c hiu

sutcao nht.

Qua sng lc ngu nhin, v t bin bng UV v tc nhn ha hc thu

c bin chng B3.04l bin chng c hot tnh khng sinh cao nht. T l


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sng st sau 3 ln t bin ca VSV l 0,15%. Sau ln men chm bin chng

B3.04 to khngsinh. Mi trng ln men thch hp nht l MT1dt. Lng

khng sinh th thu c t dch ln men l 0,1033 g/l.

Tch chit khng sinh:

Sau khi ln men, dch lc ln men c chit bng ethylacetat pH 5. Dch

chit dung mi hu c c em ct quaychn khng thu khng sinh th.

Qu trnh tch cc thnh phn trong hn hp khng sinh th cho thy hn

hp khng sinh c t nht 3 thnh phn. Cc thnh phn chnh c tinh ch

bng sc k ct Silicagel, vi hiu sut tinh ch cui cng khong 33,48%

Mt s tnh cht l ha khng sinh chnh thu c:

Nhit nng chy :217,40C.

Ph t ngoi: cho cc nh hp th : 204 nm, 333 nm v 443 nm. T ,

d on cu trc KS c nhn thm, ni i lin hp, d t, hoc kt hp cc c

im trn.

Ph khi lng phn t: d on phn t lng l 1268, 49 vC [7].

Nh vy, nghin cu bc u tin hnhsng lc, t bin thu cbin chng kh nng sinh tng hp khng sinh cao hn, v tch chit c

thnh phn khng sinh chnh trong qu trnh ln men. Tuy nhin, hiu sut sinh

tng hp khng sinh cha cao, qu trnhtinh ch khng sinh chaxc nh c

khng sinh tinh khit hay cha, do cn tip tc nghin cu nng cao kh

nng sinh tng hp,hiu sut qu trnh tinh ch, v tinh khitca khng sinh

chnh thu c.

1.3. Nghin cu tng hp Actinomycin X2 t Streptomyces 15.29

Streptomyces microfl avus

Streptomyces 15.29 l chng x khun c phn lp t t Vit Nam,

ti phng th nghim vi sinh, b mn Vi sinh sinh hc trng H Dc H Ni.


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Tin hnh phn loi Streptomyces 15.29 theo ISP th thy 92,68% cc c im

trng khp vi Streptomyces sinensis. Tuy nhin, khi gii trnh t gen 16s rADN

bng k thut khuch i gen PCR thy h gen ca Streptomyces 15.29 hon

ton trng vi gen ca Streptomyces microflavus. Do kt lun tn khoa hc

ca Streptomyces 15.29l Streptomyces microflavus.

Tin hnh ln men Streptomyces microflavus thu dch ln men sau chit

bng ethylacetat. Dch chit ethylacetat c ct chn khng thu c cn (5g),

sau tin hnh cc phng php tinh ch bng sc k lp mng v sc k ct

silica gel thu c cht khng sinh chnh1(1g).

Khng sinh chnh 1 c xc nh cc thng s v c tnh l ha v cng

thc phn t, cng thc cu to.

Khng sinh chnh 1 :Cht rn c mu hng sm, Ph khi lng ESI-MS

m/z 1292 vC [M+Na]+C62H84N12O17.

Nhit nng chy: 249 - 250oC.

quay cc: []22D-360o(c, 0,5 trong MeOH).

Tin hnh o ph cng hng t ht nhn v bin gii ph, tc gi xcnh c hp cht 1 l actinomycin X2, mt hp cht cng c phn lp t

chng Streptomycesspp. v c hot tnh chng ung th cng nh hot tnh khng

sinh rt mnh[16, 17, 20].

1.4. Cng ngh ln men Daunorubicin v Adriamycin.

Daunorubicin v Adriamycin l cc khng sinh c hot tnh chng ung th

t cc x khun chi Streptomyces. Daunorubicin c sinh tng hp t chng S.

peucetius v S.coeruleorubidus c c cao nn ngy nay khng c s dng

iu tr ung th, thay vo ngi ta nghin cu ra Adriamycin l cht khng

sinh tng hp t bin chng ca S. peucetius c hot tnh cao hn v ph iu tr

ung th rng hn, t c tnh hn. Ngoi ra, mt nhm nghin cu Lin X


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cng thu c mt dn xut tng t khc l carmynomycinsinh tng hp t

x khun Actinomadura carminata.

Qu trnh ln men sn xut Adriamycin nh sau: Chng S. peucetius c

t bin bng ha cht thu bin chng. Bin chng c nui cy trong ng

thch nghing 26-270C, trong khong 10 ngy, sau loi b h si nm c

mu m cy chuyn sang mi trng bnh tam gic 2 lt v nui trn my lc

tc 120 v/pht, 280C trong 2 ngy. Sau cy sang thit b nhn ging th

tch 170 lt ( cha 120 lt mi trng), nhn ging 26 -270C trong 27 gi, ri

cy truyn tip sang thng ln men 800 lt ( cha 500 lt MT). Qu trnh ln men

ko di khong 145 gi.

Sn phm adriamycin tn ti trong c mi trng ln hsi, v vy ngi

ta phi x l c phn sinh khi v phn dch trong thu sn phm. u tin,

dch ln men c lc tch ring dch trong v b lc. Phn b lc c ngm

x l trong hn hp dung mi aceton: acid sulfuric 0,1N t l 4:1, sau lc

tch b. Phn dch c trung ha v pH 7, v c c chn khng xung nng

Adriamycin khong 250mg/l. Dch c c c loi lipid bng trch ly vi dungmi chloroform pH 3. Pha nc c kim ha bng NaOH v pH 8,6 ri trch

ly bng h dung mi ChloroformMethanol. Dung mi trch ly loi nc c

lm kh bng natri sulfate khan, lc, c c, cui cng b sung ether vo kt

ta thu adriamycin th. Phn dch trong thu c sau khi lc dch ln men cng

c x l qua cc cng on tng t thu btadriamycin th. Bt th c ha

tan li trong nc tch qua ct cellulose, ra ct bng m phosphate pH 4,5

v n-butanol. Dch thu gom cc phn on sn phm c c c chn khng ri

trch ly sang chloroform ( t l dung mi 4:1). Bc tip theo l phn tly thu pha

dung mi, lm kh bng Natri sulphat khan, c c chn khng ri b sung dung

dch HCl trong methanol v lm lnh kt tinh thu kt ta. Cui cng, ha tan


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v kt tinh li kt ta trong ethanol thu sn phm Adriamycin hydrochlorid

tinh sch.[2, 8].

1.5. Tng hpcirramycin B tStreptomyces cyaneus

Atta v cng s tin hnh nghin cu lnmen sinh tng hp v tinh ch

cirramycin B t S. Cyaneus

Sau khi ln men, dch lc ln men c chit xut bng Ethylacetat t l

1:1. Loi dung mi bng ct chn khng di p sut gim thu c cn, cn

c ha tan trong lng ti thiu DMSO, sau c lc. Cc thnh phn

khng sinh c tch bng sc k lp mng vi h dung mi chy sc k l

Chloroform: Methanol ( 24:1). C t nht 3 thnh phn trong hn hp khng

sinh, trong thnh phn c Rf= 0,4 l thnh phn chnh.

Tinh ch khng sinh chnh bng sc k ct silicagel vi dung mi chy ct

l Chloroform: methanol (9:1).

Khng sinh chnh thu c c nhit nng chy l 2280C, ha tan khng

gii hn trong CHCl3, ethylacetat, n butanol, aceton, ethanol, methanol, iso-

propanol, khng tan trong du m, hexan, benzene. o ph UV, bc sng hpthu cc i l 240nm, ph IR c 11 nh hp th. o ph khi xc nh khi

lng phn t l 679,4 vC, cng thc phn t l C36H59NO12.

Tin hnh phn tch ph cng hng t ht nhn v bin gii ph xc

nh c khng sinh chnh l cirramycin mt khng sinh th h II nhm

macrolide, c kh nng c ch c vi khun Gr+ v Gr-[20].

1.6. Khng sinh chng nm non-polyen tStreptomyces albidoflavus

Khng sinh chng nm ch chim mt t l rt nh trong cc nhm thuc

hin nay , tuy nhin li ng vai tr rt quan trng trong vic kim sot cc bnh

v nm.


17/92

10

S K. Augustine v cng s tin hnh nghin cu tng hp mt khng

sinh chng nm non polyen t Streptomyces albidoflavus PU23.

Lng khng sinh thu c t Streptomyces albidoflavus PU23cao nht

l vo ngy th 8 ca qu trnh nui cy. Dch ln men c chit bng n-hexan,

sau bc hi thu hi dung mi thu c khng sinh th. Cc thnh phn trong

khng sinh th ny c phn tch bng sc k lp mng vi h dung mi l

ethanol: nc:chloroform (2:2:1). Dng sc k ct vi h dung mi l ethanol:

nc ( 50:50) tinh ch thu khng sinh tinh khit chnh.

Da vo phn tch ph FTIR, ph cng hng t ht nhn d on cng

thc phn t ca khng sinh chnh l C16H32O19, c mt nhm hydroxymethyl,

mt ni i, cn li l cc lin kt CHOH-. Nh vy khng sinh chnh c cng

thc cu to kiu chui polyhydroxy polyether mch thng, c mt lin kt i ,

do thuc nhm chng nm non-polyen [18].

V hot tnh sinh hc ca khng sinh chnh i vi cc chng nm c

th hin bng di y:

Bng 1.1 : Gi tr MIC v MFC ca khng sinh t S.albidoflavus.

1.7. Tng hp khng sinh antimycin A9 tStr eptomyces Sp. K01-0031


18/92

11

Chng K01-0031 c phn lp t mu t trn ni ti Chuxiong,

Yunnan, Trung Quc. Qua phn loi ISP, xc nh c l thuc nhm x khun

Streptomyces.

Tin hnh ln men chng x khun trn, dch ln men c chit bng

ethylacetat, sau ct thu hi dung mi thu c cn. Tin hnh sc k ct hn

hp khng sinh th, thu c hn hp khng sinhnhm antimycin. Hn hp ny

c chy qua sc k lng hiu nng cao HPLC, dung mi ra gii l CH 3CN

70% thu c khng sinh tinh khit chnh l antimycin A9(1). Bn cnh cn

thu c antimycin A3a (2), A3b(3), A4(4),A7(5).Antimycin A9 l khng sinh chnh, n tan c trong MeOH, aceton,

acetonitril, ethyl acetat v chloroform, khng tan trong nc v n-hexan. Cng

thc ca antimycin A9 c xc nh l C29H34N2O9 . Ph IR ca antimycin A9

tng t nh ph ca mt s antimycin bit. Phn tch ph 1H, 13C NMR

thy cng thc cu to ca (1) c mt vng dilactone, mt nhm bn 3-

formamidosalicyl, mt chui n-butyl. Nhng c im ny ging vi cng thc

cu to ca cc antimycin khc. Tuy nhin, (1) l cht duy nht c nhm

phenylacetyl gn vi C8[25].

V kh nng khng khun, nghin cu nh gi trn hai chng vi khun

Caenorhabditis elegans, vArtemia salina. Kt qu c th hin trn bng sau:

Bng 1.2: Gi tr MIC ca cc antimycin trn Caenorhabdit is elegans, v

Ar temia salina

Hp cht Caenorhabditis elegans Artemia salina

Antimycin A9 0.2 0.05

Antimycin A3a 1 0.2

Antimycin A3b 1 0.2


19/92

12

Antimycin A4 1 0.2

Antimycin A7 >1 0.2

1.8. Sparsomycin khng sinh c tng hp tStreptomyces Sp. AZ-

NIOFD1

Atta v cng s tin hnh phn lp mt chng x khun Streptomyces

sp.AZ-NIOFD1 t nc sng Nin-Ai Cp, sau tin hnh ln men, chit xut

v tinh ch thu c mt hp cht c hot tnh khng sinh.

Tin hnh gii trnh t gen 16s rADN ng dng k thut khuch i gen

PCR nhn thy Streptomyces sp. AZ-NIOFD1 tng t Streptomyces

violaceusniger ,AZ-NIOFD1.

Dch ln men c chit bng n-butanol pH 7, ct chn khng loi dung

mi. S dng sc k lp mng tch cc thnh phn trong hn hp khng sinh

th, dung mi l chloroform : methanol (24:1), ch cht c Rf= 0.75 l c hot

tnh khng sinh. Tin hnh tinh ch bng sc k ct silica gel vi h dung mi

chy ct l chloroform : methanol (8:2), phn on t 23 n 30 l c hot tnhkhng sinh mnh nht.

Khng sinh chnh tinh khit c xc nh cc thng s ha l v cu

trc. Khng sinh chnh tan c trong chloroform, n-buthanol, acetone,CCl4,

methanol, ethanol, DMSO, khng tan trong du ha, hexan, v benzen. Nhit

nng chy l 2050C. Cng thc phn t l C13H21N3O6S2, phn t lng l 361.3

vC, ph IR c 23 nh hp th. Ph UV hp th cc i 270 v 302 nm. Phn

tch ph cng hng t kt hp vi cc thng s trn c th kt lun khng sinh

chnh sinh tng hp t Streptomyces Sp. AZ-NIOFD1 l sparsomycin.


20/92

13

Nghin cu hot tnh sinh hc ca khng sinh ny thy n c kh nng c

ch Gram dng ( MIC dao ng t 11,7 n 31,25 g/ml), Gram m ( MIC dao

ng t 11,7 n 15,6 g/ml) [19].


21/92

14

CHNG2. I TNG V PHNG PHP NGHIN CU

2.1. Nguyn vt liu v thit b

2.1.1. Nguyn vt liu Chng x khun:chng Streptomyces 173.113do B mn Vi sinh Sinh

hc trng i hc Dc H Ni phn lp t mu t ti Ty Nguyn.

Chng vi sinh vt kim nh:do B mn Vi sinh Sinh hc cung cp, c

trnh by bng 2.1.

Bng 2.1: Cc vi khun kim nhVi khun Gram(+) Vi khun Gram(-)

Staphylococcus aureus ATCC 1228 Proteus mirabilis BV 108

Mi trng

Cc mi trng nui cy x khun: Theo nghin cu trc [7], chng ti

la chn MT1 lm mi trng nui cy x khun vi thnh phn trong 100 ml

mi trng nh sau: Tinh bt 2g, KNO30,1 g, K2HPO4

0,05g, MgSO4.7H2O 0,5

g, NaCl 0,5 g, thch 1,8 g, pH 7.

Cc MT ln men (MTdt): C thnh phn tng ng MT nui cy x khunnhng loi b thch.

Cc MT nui cy VSV kim nh: thnh phn cc MT c trnh by

bng 2.2.

Ch : Cc MT nui cy x khun, MT ln men v MT nui cy VSV kim nh

u c tit trng 118-120C trong 30 pht


22/92

15

Bng 2.2: Cc MT nui cy VSV kim nh

Thnh phnNaCl Cao tht Pepton Thch

pH

% % % %

MT canh thang 0,5 0,3 0,5 07,0-7,4

MT thch thng 0,5 0,3 0,5 1,8

Dung mi

Bng 2.4 gii thiu cc dung mi c s dng trong nghin cu cng cc

c trng c bn ca chng.Bng2.3: Cc dung mi s dng

Dung mi Khi lng ring (g/ml) Nhit si (C)

Aceton 0,79 56

Ethanol 0,789-0,791 78,3

Ethylacetat 0,889-0,901 70,4

Methanol 0,791-0,793 88,6-88,8Triethylamin 0,726-0,728 90

n-hexan 0,655 68,7

n-propanol 0,803 97-98

Vt liu chy sc k

- Bn mng sc k: Silicagel 60 F254, Merck.

- Dung mi chy sc k: Cc dung mi trong bng 5 trn

- Bnh chy sc k, buret, Silicagel 60 F254 ht c ng knh 0,04-0,063

mm


23/92

16

2.1.2. My mc thit b- Tc nhn gy t bin: nh sng UV (=254nm), ngun pht Toshiba

60W, in th 220V.

- My lc Taitec Bio Shaker BR 300 Lf

- T m Memmert, Binder

- T cy v trng Aura VF 48

- T sy SL shellab

- T lnh Sanyo

-Ni hp v trng Hirayama Model HL3030

- Cn k thut Sartorius TE 210- Cn phn tch Sartorius BP 121 S

- My o nhit nng chy E2-Melt

- My ct quay Buchi Waterbath B480

- Thc kp Palmer chnh xc 0,02mm

- Hp Petri ng knh 9cm

- Buret, bnh chit, patuyn, pipet cc loi, ng nghim, bnh nn, ng

ong, cc c m cc loi, nt thy tinh, bng gc, giy lc, que cy, giy th pH,

n cn, a thy tinh, kim mi mc,

2.2. Phng php thc nghim

2.2.1. Nui cy v gi ging x khun

- Cy zigzac x khun trn ng thch nghing c cha MT nui cy thch

hp, cho pht trin 28-29C.

- Sau 6-10 ngy, x khun pht trin tt, cho cc ng ging vo t lnh

bo qun 2-3C, nh k 3-6 thng cy chuyn.


24/92

17

2.2.2. nh gi hot tnh khng sinh bng phng php khuch tn

- Nguyn tc:Mu th c t trn lp thch dinh dng cy VSV

kim nh, khng sinh t mu th khuch tn vo MT thch s c ch s pht

trin caVSV kim nh to thnh vng v khun.

- Tin hnh:

+ To hn dch VSV: Ly mt vng que cy VSV kim nh cy vo 2.5

ml MT canh thang, 37C trong 18-24h (i vi vi khun) to thnh hn

dch VSV c nng 106-108t bo/ml.

+ Cy hn dch VSV vo mi trng thch thng tit trng v

ngui n 45-50C vi t l 2,5:100 (105 t bo/1ml), lc u, vo cc a

Petri v trng vi th tch 20ml/a.

+ a mu th vo mi trng kim nh theo 1 trong 3 cch sau:

Phng php khoanh giy lc: Khoanh giy lc (=6,0mm)

c tm 3 ln dch chit dung mi hu c ca mu th, sy kh nhit di

500C, sau t ln b mt mi trng cy VSV kim nh.

Phng php khi thch: t khi thch (=6,0mm) c cha VSVsinh khng sinh ln b mt MT cy VSV kim nh.

Phng php ging thch: c cc ging thch (=6,0mm) trn

MT cy VSV kim nh. Nh 0,05ml dch th vo mi ging.

+ Cc a Petri c mu th c trong t m 37C trong 18 -24h (i

vi vi khun). Mi mu c th song song trn 3 a vi cng 1 loi VSV kim

nh.+ nh gi kt qu: o ng knh vng v khun bng thc kp Palmer

c chnh xc 0,02mm. Kt qu c nh gi da trn ng knh vng v

khun x l theo cng thc:


25/92

18

2

1 1

( )

1

n n

i i

i i

D D D

D sn n

Trong :

D (mm): ng knh trung bnh vng v khun

iD (mm): ng knh vng v khun th i

s: lch thc nghim chun c hiu chnh

n: S thc nghim tin hnh song song (Thng thng n=3)

2.2.3. Sng lc ngu nhin

- Pha hn dch bo t: Ly mt vng que cy bo t Streptomyces

173.113,cho vo ng nghim cha 10ml nc ct v trng, lc cho bo t phn

tn u, thu c hn dch bo t pha long 10 -1(nh c). Sau , pha

long tng t n nng 10-6bng nc v trng.

- Cy bo t: Dng pipet v trng ht chnh xc 0,1ml hn dch bo t

cc pha long 10-4, 10-5, 10-6, nh ln b mt thch ca MT2 trong a Petri.

Dng que trang v trng dn u hn dch bo t ln b mt thch. Tin hnh

trn 3 a Petri cho mi nng pha long. Cho cc a vo t m 28C trong 6

ngy cho xut hin cc khun lc mc ring r.

- Php chn lc ngu nhin: Chn ngu nhin cc khun lc pht trin c

th, mc ring r sau 6 ngy ri ly ra cy th HTKS. Cc khun lc ny, sau khi

nui cy 6 ngy c th hot tnh khng sinhbng phng php khi thch.

2.2.4. t bin bng nh sng UV

- Pha hn dch bo t: Ly mt vng que cy bo t Streptomyces

173.113,cho vo ng nghim cha 10ml nc ct v trng,lc cho bo t phn

tn u, thu c hn dch bo t pha long 10 -1(nh c). Sau dung


26/92

19

1ml hn dch bo t ny pha long n nng 10-6lm mu chng, 9ml cn li

c a vo a Petri v trng.

- t bin di tc dng ca nh sng UV: 9mlhn dch bo t nng

10-1 ng trong a Petri c chiu nh sng UV (=254nm) vi khong cch

v thi gian thch hp (khong cch 60cm, thi gian 5 pht), sau ly ra,

ch ti 2h, ri dng pipet v trng pha long n nng 10-5bng nc v

trng.

- Cy cc bo t sau t bin: Dng pipet v trng ht chnh xc 0,1ml

mu chng 10-6v 0,1ml hn dch t bin cc nng 10-4v 10-5vo cc

a Petri c cha MT1, sau dng que trang v trng dn u git hn dch bo

t trn b mt thch, mi pha long lm 3 a song song. Cc a Petri sau khi

cy c nhit 28C trong 6 ngy n khi xut hin khun lc. m cc

khun lc, xc nh % sng st theo cng thc:

% sng st = mo

N

Nx 10-6+kx 100%

Trong : Nm: S khun lc sau t bin nng 10-k

No: S khun lc trong mu chng nng 10-6.

- Tin hnh sng lc vi cc khun lc sau khi t bin tng t nh

phng php sng lc ngu nhin. Lm song song mu chng. Phn trm bin

i hot tnh c xc nh theo cng thc:

% bin i hot tnh = 100%io

D

D

Trong :iD : ng knh trung bnh vng v khun ca bin chng th i sau t

bin

oD : ng knh trung bnh vng v khun ca mu chng.


27/92

20

- Sau khi tin hnh t bin, da vo kt qu thu c la chn ra cc

bin chng c % bin i hot tnh (+) cao nht dng cho cc nghin cu tip

theo.

2.2.5. Ln men chm tng hp khng sinh

-Nguyn tc:Sau khi c kt qu xc nh c cc mi trng nui cy

b mt tt nhtcho vic sn xut khng sinh, s dng ging cp 1 ln men

gin on trong cc mi trng lng tng ng.

- Tin hnh

+ To ging cp 1: Chng ging Streptomyces 173.113sau khi nui cytrn thch nghing (MT1) 6 ngy tui c cy v trng sang bnh nn 500ml

cha 100ml MT1dt bng 10ml nc v trng, lc trn my lc trn nhit

28C 0,1C, tc quay 140 vng/pht trong 48h.

+ Ln men: Sau 48h nhn ging, tin hnh ln men trn mt sMT khc

nhau chn MT ln men tt nht. Ging cp 1 c cy v trng sang bnh nn

500ml cha cc MT dinh dng vi t l Vging:Vmi trng= 1:10. Cc bnh ln

men c lc nhit 28C 0,1C, tc quay 140 vng/pht trong 120h.

chn chng c kh nng ln men sinh tng hp khng sinh tt nht:

ging cp 1 trn MT1dt c ln men trn mi trng ti thch cho vic sinh

tng hp khng sinh, sau la chn chng cho dch ln men c HTKS tt nht.

2.2.6. Chit khng sinh t dch ln men bng dung mi hu c

-Mc ch:Xc nh dung mi v pH chit ti u cho dch lc.- Tin hnh:Dng phng php chit 1 ln.

+ Dch lc c chnh v cc pH 3, 5, 7, 9, 11 bng dung dch NaOH 1M

v HCl 1M.


28/92

21

+ Chit dch lc chnhpH vi nhiu dung mi khc nhau: Dch lc v

dung mi cho vo bnh gn theo t l Vdung mi:Vdch lc= 1:5. Sau lc k trong

1 pht. yn 1h cho phn lp. Tch ring ly lp dung mi v dch lc.

+ Sau mi ln chit, ly lp dung mi v lp dch lc th hot tnh khng

sinh theo phng php khoanh giy.

2.2.7. Tch cc thnh phn trong khng sinh bng sc k lp mng

-Mc ch:Xc nh cc thnh phn khng sinh trong dch chit, dch lc

hoc xc nh khng sinh tinh khit hay cha.

- Tin hnh:+ Ct bn mng c kch thc thch hp, hot ha 110C/30 pht.

+ Pha hn hp dung mi theo ng t l, cho vo bnh sc k (lp dung

mi khng qu 1cm), yn bnh khong 1h bo ha dung mi.

+ Dng ng mao qun ng knh 0,5mm chm dung dch cn sc k ln

bn mng. Chm nhiu ln, kh t nhin hoc sy 50C.

+ t bn mng vo bnh sc k. Cho dung mi chy ln khong chiu

di bn mng th ly ra, nh du ng dung mi chy.

+ Xc nh vt theo cc phng php sau:

Pht hin bng mt thng.

Soi n t ngoi: t bn mng sc k vo n t ngoi, vt cht

th s hin mu.

Phng php hin hnh VSV: t bn mng vo a Petri tit

trng, mt lp va phi MT thch thng cy VSV kim nh sao cho bn

mng chm trong thch. vo t 37C, trong 18-24h, vng v khun xut

hin nu c khng sinh.


29/92

22

Phng php hin mu ha hc: Bn mng c phun thuc th

hin mu, sy 70C trong 10 pht hin vt. Dng thuc th ninhydrin 1%

trong cn pht hin cc khng sinh c cha nhm amin.

+ Tnh h s Rf(Retardation factor)

f

aR

b

Trong : a: Khong cch t vch xut pht n tm ca vt hat cht

b: Khong cch t vch xut pht n vch dung mi.

2.2.8. Thu khng sinh th bng phng php ct quaychn khng

Pha dung mi hu c thu c sau khi chit c ct quay bng my ct

chn khng p sut -0,09 atm. Cn thu c c ha tan bng mt lng nh

methanol hoc aceton, vo bnh nn sch, bc hi 50C n kh. Co, thu

ly bt khng sinh th.

2.2.9. Tinh ch khng sinh th bng sc k ct

- Pha h dung mi chy theo ng t l, 30 pht cho bo ha.- Chun b ct sc k: Ct c ng knh 1cm, di 50cm c ra sch,

trng cn, sy kh. Cn khong 8g ht Silicagel nhi ct, hot ha 110C trong

30 pht, em ha thnh hn dch vi dung mi chy v a ln ct.

- a mu th ln ct: Bt khng sinh th ha tan trong mt lng ti

thiu methanol ri trn vi khong 2g Silicagel hot ha. Sy 50C cho bay

ht methanol ri cho ln ct,n nh ct trong 30 pht.

- Cho dung mi chy qua ct vi tc 0,5ml/pht. Ly cc phn on

vo ng nghim sch, khong 30 phn on, mi phn on 5ml.

- Pht hin v xc nh cc phn on cha khng sinh cn tch: Th

HTKS ca cc phn on bng phng php khoanh giy lc. Cc phn on c


30/92

23

HTKS c tin hnh sc k lp mng vi h dung mi tch tt nht. Cc phn

on chnh l cc phn on t khi xut hin vt n khi khng cn vt c Rf

tng ng v vn c hot tnh khng sinh mnh.

- Cc phn on chnh sau khi chy ct cthu vo bnh cu 250 ml, ct

quay chn khng loi dung mi thu c cn. Co thu bt tinh th khng sinh

vo ng nghim sch. Sau , bt tinh th khng sinh c kt tinh li bng hn

hp dung mi, lc, sy kh 50C thu khng sinh tinh khit.

2.2.10. Xc nh tnh cht, cu trc ha hc thnh khng sinh chnh tinh

khit thu c

- o nhit nng chy.

- Ph hng ngoi.

- Ph t ngoi.

- Ph khi MS.

- Ph cng hng t ht nhn: 1H NMR, 13C NMR, DEPT, COSY, HSQC,HMBC.


31/92

24

CHNG3: KT QU THC NGHIM V NHN XT


3.1.1. Kt qu sng lc ngu nhin

K tha kt qu ca nghin cu trc [7] chng ti la chn vi khun

Staphylococcus aureus vProteus mirabilisl 2 chng VSV kim nh i din

thc hin cc nghin cu v sau. Tin hnh sng lc ngu nhin trn chng

Streptomyces 173.113. B3.04l chng tt nht t nghin cu trc ang c

lu gi ti b mn Vi sinh sinh hc, trng H Dc H Ni.

* Mc ch:Chn dng chng c HTKS cao trong qun th.

* Tin hnh v kt qu:

Chng Streptomyces 173.113.B3.04 c SLNN, gi li 32 dng chng,

th HTKS bng phng php khi thch. Kt qu c th hin trn bng 3.1.

Bng 3.1: Kt qu th HTKS sng lc ngu nhin

Dngchng

Hot tnh khng sinh Dngchng

Hot tnh khng sinhP. mirabilis S. aureus P. mirabilis S. aureusD

(mm) sD

(mm) SD

(mm) SD

(mm) s

SL.01 17,97 1,23 20,05 0,39 SL.17 14,59 1,19 19,31 0,33

SL.02 19,23 0,51 21,45 0,64 SL.18 17,42 0,41 19,50 0,37

SL.03 16,02 0,97 20,46 1,41 SL.19 16,87 1,98 20,65 1,15

SL.04 16,64 1,17 20,73 0,88 SL.20 16,07 1,16 19,31 0,90

SL.05 18,02 0,53 19,97 1,10 SL.21 16,20 0,59 18,99 0,80

SL.06 17,04 0,82 20,27 1,63 SL.22 14,45 0,97 19,47 0,37

SL.07 18,63 0,44 21,29 0,77 SL.23 16,61 1,02 19,55 1,10

SL.08 18,76 0,52 21,01 0,40 SL.24 15,27 0,94 19,23 0,50

SL.09 19,35 0,55 21,37 0,23 SL.26 17,95 0,36 19,05 0,37

SL.11 16,11 2,24 20,50 0,40 SL.27 18,29 0,64 19,69 1,46

SL.12 18,66 0,63 21,18 0,45 SL.28 18,95 0,99 20,93 0,93

SL.13 19,39 0,55 21,51 0,26 SL.29 18,65 0,47 20,89 0,25


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SL.14 14,89 1,60 19,71 0,39 SL.30 17,27 0,49 19,49 0,73

SL.15 17,01 0,84 19,63 0,73 SL.31 18,47 0,54 20,58 0,35

SL.16 16,33 0,71 19,13 0,20 SL.32 18,29 1,51 20,45 0,49

* Nhn xt:

Theo bng 3.1 c th thy 3 dng chng SL.02, SL.09 v SL.13l nhng

dng chng c hot tnh khng sinh cao nht. Do , gi 3 dng chng ny li

cho nghin cu tip theo.

3.1.2. Kt qu t bin ln th nht

* Mc ch:

S dng nh sng UV gy t bin nhm tng hot tnh khng sinh ca

chng x khun thu c sau SLNN.

* Tin hnh v kt qu:

Chn dng chng c HTKS cao nht sau SLNN l SL.13, mang t bin

bng nh sng UV (=254nm), khong cch chiu 60 cm, thi gian 5 pht.

T l sng st l 0,73%, cch tnh c th hin trong bng 3.2.

Bng 3.2. Tnh ton t l sng st sau t bin ln th nht

a ix (S khun lc trung

bnh)

s

Sau t bin nng 10-422,67 5,13

Sau t bin nng 10-56,33 1,53

Chng nng 10-6116,33 5,51

T l sng st 0,73%Chn v nui cy 32 bin chng, th HTKS bng phng php khi

thch. Kt qu c th hin bng 3.3.


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Bng 3.3. Kt qu th HTKS sau t bin ln th nht

K hiu

binchng

Hot tnh khng sinh

P. mirabilis S. aureusD

(mm)s

% hottnh so vi

MC

D (mm)

s% hot

tnh so viMC

MC 21,02 0,09 100,00 23,08 2,31 100,00

B1.01 20,87 1,11 99,30 22,79 0,96 103,20

B1.02 22,45 0,77 106,82 23,28 0,87 105,43

B1.03 21,21 0,66 100,92 23,21 1,11 105,13

B1.04 23,09 0,05 109,86 24,01 0,93 108,76

B1.05 22,27 0,93 105,96 22,86 0,71 103,53

B1.06 22,25 1,17 105,87 19,52 2,17 88,41

B1.07 19,46 0,75 92,58 22,36 2,13 101,27

B1.08 20,51 1,89 97,56 22,13 0,55 100,24

B1.09 22,10 1,21 105,14 23,12 0,87 104,71

B1.10 19,71 1,67 93,78 20,61 0,68 93,33

B1.11 21,45 0,88 102,06 23,01 0,37 104,23

B1.12 22,39 2,92 106,50 23,75 0,99 107,55

B1.13 23,81 0,37 113,26 24,15 0,26 109,39

B1.14 21,41 0,28 101,84 23,11 0,80 104,68

B1.15 21,87 0,88 104,03 23,75 0,10 107,58

B1.16 22,59 0,12 107,48 21,98 0,64 99,55

B1.17 20,25 0,41 96,35 21,32 0,57 96,56B1.18 21,87 0,29 104,06 22,88 0,53 103,62

B1.19 22,46 0,61 106,85 20,89 0,85 94,63

B1.20 22,56 1,00 107,33 23,89 0,18 108,18

B1.21 20,45 3,86 97,30 23,29 0,44 105,50


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B1.22 19,60 1,28 93,24 22,31 1,50 101,03

B1.23 21,16 1,53 100,67 22,61 1,06 102,42

B1.24 18,44 0,96 87,73 20,71 0,73 93,78B1.25 18,69 0,55 88,90 21,39 0,59 96,86

B1.26 18,93 0,53 90,07 21,21 0,40 96,04

B1.27 20,11 0,85 95,65 20,86 1,07 94,47

B1.28 21,17 0,69 100,73 20,57 1,44 93,18

B1.29 22,10 0,78 105,14 22,87 0,81 103,56

B1.30 23,57 0,29 112,15 24,28 0,18 109,98

B1.31 19,29 0,87 91,79 22,29 0,50 100,94B1.32 21,89 0,97 104,12 23,91 1,00 108,30

* Nhn xt:

Sau khi t bin bng nh sng UV vi t l sng st l 0,73%th hot

tnh khng sinh ca chng x khun tng ln ng k trn cP. mirabilis v S.

aureus, nht l cc bin chng B1.04, B1.13 vB1.30. Do , tin hnh

nui cy v gi ging 3 bin chng ny. La chn bin chng B1.13 tip

tc t bin ln 2.

3.1.3. Kt qu t bin ln th hai

* Mc ch:

Tip tc s dng nh sng UV gy t bin nhm tng hot tnh khng

sinh ca chng x khun thu c sau t bin ln th nht.

* Tin hnh v kt qu:

Chn dng chng c HTKS cao nht sau t bin ln th nht l B1.13,

mang t bin bng nh sng UV (=254nm), khong cch chiu 60 cm, thi

gian 4 pht.

T l sng st l 0,58%, cch tnh c th hin trong bng 3.4.


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Chn v nui cy 32 bin chng, th HTKS bng phng php khi

thch. Kt qu c th hin bng 3.4.

Bng 3.4.Tnh ton t l sng st sau t bin ln th hai

a ix (S khun lc trung bnh) s

Sau t bin nng 10-420,67 9,87

Sau t bin nng 10-55,33 3,21

Chng nng 10-6127 42,53

T l sng st 0,58%

Bng 3.5. Kt qu th HTKS sau t bin ln th hai

K hiubinchng

Hot tnh khng sinhP. mirabilis S. aureus

D (mm)

s% hot

tnh so viMC

D (mm)

s% hot

tnh so viMC

MC 22,62 0,56 100,00 23,38 0,58 100,00

B2.01 23,01 0,74 101,74 23,85 0,63 102,02B2.02 24,94 0,77 110,26 26,03 0,07 111,32B2.03 23,05 1,14 101,92 24,21 0,96 103,54B2.04 23,09 0,05 102,09 24,01 0,93 102,71B2.05 23,27 0,60 102,89 23,95 0,45 102,45B2.06 22,25 1,17 98,38 23,08 1,92 98,72B2.07 21,13 2,65 93,43 22,38 2,57 95,72B2.08 22,90 1,54 101,24 23,42 0,75 100,17

B2.09 23,33 0,60 103,15 24,11 0,79 103,14B2.10 24,56 0,43 108,58 25,01 0,02 106,96B2.11 23,05 1,50 101,92 23,59 0,52 100,88B2.12 22,39 0,97 98,97 24,65 0,34 105,45B2.13 23,81 0,37 105,25 24,15 0,26 103,31B2.14 22,39 1,11 98,97 25,16 0,82 107,61


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B2.15 23,01 0,87 101,71 23,75 0,10 101,60B2.16 22,59 0,12 99,88 24,48 1,56 104,70B2.17 24,35 0,91 107,65 21,47 0,69 91,84

B2.18 22,21 0,49 98,17 22,88 0,53 97,86B2.19 22,95 0,95 101,44 20,93 0,90 89,51B2.20 23,39 0,87 103,42 24,19 0,59 103,45B2.21 22,50 0,97 99,47 23,58 0,51 100,86B2.22 23,34 1,80 103,18 22,59 1,86 96,64B2.23 22,99 1,13 101,65 22,26 1,03 95,21B2.24 21,84 2,16 96,55 20,71 0,73 88,57

B2.25 24,60 0,63 108,75 25,35 0,37 108,41

B2.26 20,67 1,92 91,36 21,21 0,40 90,70B2.27 21,91 1,86 96,88 20,86 1,07 89,22B2.28 24,37 0,46 107,75 20,57 1,44 88,00B2.29 22,10 0,78 97,70 22,87 0,81 97,80B2.30 23,77 0,45 105,10 24,28 0,18 103,86B2.31 20,97 2,98 92,72 22,29 0,50 95,32B2.32 22,79 1,28 100,74 23,91 1,00 102,28

* Nhn xt:

Sau khi t bin bng nh sng UV vi t l sng st l 0,58%th hot

tnh khng sinh ca chng x khun tng ln ng k trn cP. mirabilis v S.

aureus, nht l cc bin chng B2.02, B2.10vB2.25. Do , tin hnh

nui cy v gi ging 3 bin chng ny.

3.2. Xc nh iu kin ln men, chit tch khng sinh tt nht

3.2.1. Kt qu chn chng ln men

K tha kt qu ca nghin cu trc[7], chng ti bit MT1dt l mi

trng ln men chm tt hn cc mi trng cn li i vi chng Streptomyces


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173.113. Do , tip tc tin hnh chn chng ln men chm tt nht trn

MT1dt.

* Mc ch:

Chn c bin chng (hoc dng chng) c kh nng ln men chm tt

nht t cc bin chng, dng chng c lu gi qua cc th nghim sng lc

ngunhin, t bin ln 1, ln 2.

* Tin hnh v kt qu:

Thc hin ln men chm ring r cc bin chng ny trong MT1dt. Dch

ln men c lc qua giy lc loi sinh khi ri th HTKS dch lc bng

phng php ging thch. Kt qu c trnh by bng 3.6.

Bng 3.6. Kt qu chn chng ln men

Dng chng, bin chngHot tnh khng sinh

P. mirabilis S. aureus

D (mm) s D (mm) s

SL.02 19,06 0,70 20,83 0,93

SL.09 19,23 0,51 21,63 0,42

SL.13 20,07 0,08 21,31 0,56

B1.04 23,06 0,65 24,49 0,58B1.13 23,85 0,79 24,81 0,38B1.30 23,52 0,46 23,53 0,53

B2.02 25,06 0,20 26,36 0,48B2.10 24,71 0,38 24,37 0,61B2.25 24,50 0,62 25,61 0,52

*Nhn xt:Trong 9 dng chng (bin chng) em ln men chm th dch lc ca dch

ln men chng B2.02c HTKS cao nht. Do , chn chng B2.02 ln

men to khng sinh.


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3.2.2. Kho st chn mi trng ln men chm

T kt qu ca nghin cu trc [7], thy MT1dt c kh nng ln men

chm tt hnso vi cc mi trng ln men khc. Nay, chng ti tin hnh kho

st nng cao kh nng ln men ca MT1dt bng cch cho thm cao nm men

vi cc t l 0,1%, 0,5%, 1%, 2% c k hiu ln lt l MT1dt.1; MT1dt.2;

MT1dt.3; MT1dt.4 t tm ra t l tt nht lm mi trng ln men chm

cho cc nghin cu v sau.

Kt qu kho st c trnh by bng 3.7.

Bng 3.7. Kt qu chn mi trng ln men

K hiumi

trng

Hot tnh khng sinh

P. mirabilis S. aureus

D (mm)

s% hot

tnh so viMTdt1

D (mm)

s% hot

tnh so viMTdt1

MT1dt 25,50 0,45 100,00 26,65 0,43 100,00

MT1dt.1 25,53 0,57 100,13 26,84 0,85 100,71

MT1dt.2 27,33 0,42 107,16 28,43 0,64 106,69

MT1dt.3 26,95 0,17 105,67 27,77 0,89 104,22

MT1dt.4 25,31 1,71 99,27 26,32 0,52 98,76

Nhn xt: Khi thm 0,1% cao nm men vo MT1dt hot tnh khng sinh

khng thay i, khi thm 0.5% cao nm men vo thhot tnh khng sinh tng r

rt. Vi t l cao nm men 1% hot tnh khng sinh cng tng nhng khng bng

mi trng 0,5%, cn mi trng 2% cao nm men th li lm gim hot tnh

khng sinh ca chng ln men. Do , MT1dt.2vi t l cao nm men c

thm vo l 0,5% c la chn lm mi trng ln men cho cc m ln men

chm sau.


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3.2.3. Kt qu ln men, chit v ct quay thu khng sinh th

Tin hnh ln men chm bin chng B2.02trn MTdt1.2, lc qua giy

lc loi sinh khi, thu c 7,5 lt dch lc.

Chit 7,5 lt dch lc ny bng 1,5 ltEthylacetatth thu c 1,1 ltdch

chit khng sinh trong Ethylacetat.

Tin hnh ct quay 1,3 lt dch chit ny thu c 0,9134 gkhng sinh th

c mu nu.

Hm lng khng sinh th trong dch ln men chm = 0,1218 g/l.

Lng khng sinh th ny c gi li nghin cu v tin hnh tinh

ch.

3.2.4. Kt qu tinh ch khng sinh ln th nht

* Mc ch:

Loi tp, bc u tch cc thnh phn khng sinh khc nhau t bt khng

sinh th.

* Tin hnh v kt qu:

Theo kt qu ca nghin cu trc[7], chn h dung mi chy ct sc k

l butylacetat: aceton: triethylamin (1:2:1) nhm loi tp v bc u tch cc

thnh phn khng sinh t bt khng sinh th.

Bt khng sinh th chy qua ct sc k silicagel, h dung mi khai trin

l h dung mi : Butylacetat: Aceton: Triethylamin (1:2:1).

Thu mi phn on 5ml vo mt ng nghim sch. Nhn thy khi thu

c 15phn on th dch chy ra t ct ch cn mu vng rt nht nn kt thc

chy ct, lc ny trn ct silicagel cnlu li mt hp cht mu nu.

Th hot tnh khng sinh tng phn on bng phng php khoanh giy

lc vi chng vi sinh vt kim nh lP. mirabilis.


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Kt qu chy sc k lp mng v kt qu th HTKS mi phn on c

th hin trong bng 3.8 v bng 3.9.

Bng 3.8. Kt qu sc k ct ln th nht

Phn on D (mm) s

121,91 0,12

222,51 0,69

321,87 0,49

421,97 0,29

5

17,87 0,46617,45 1,23

716,45 1,02

810,06 0,81

99,24 0,72

1010,25 0,96

1110,13 0,28

128,19 0,89

137,93 1,46

149,06 0,51

150,00 0,00

Thc hin chy sc k lp mng mi phn on thu c vi h dung mi

Butylacetat: Aceton: Triethylamin (1:2:1). o Rftng ng vi tng phn on.


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Bng 3.9. Kt qu sc k lp mng cc phn on

Phn on Vt 1 Vt 2 Vt 3

Rf RfRf

10,53 0,45

-

20,54 0,44

-

30,51 0,44

-

40,54 0,45

-

50,54 0,46

-

6

0,54 0,45

-

70,54 0,46

-

80,54 0,44

-

90,55 0,47

-

100,53 0,44

-

110,53 0,46

-

12- 0,46 0,36

13- 0,47 0,36

14- 0,44 0,35

15- -

-

*nh gi cm quan: C mt cht mu nu c lu gi trn ct ( cht ny c

th l tp trong dch ln men b gi li ct). Cc phn on t 1-7 c mu cam

m, t phn on 8 -11 c mu vng cam nht hn, v phn on 12-15 c muvng chanh nht.

* Nhn xt:

Nhn vo bng kt qu 3.8, c th thy phn on 15 khng c hot tnh

khng sinh. Cc phn on cn li u c hot tnh khng sinh trong phn


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on t 1 n 7 c hot tnh khng sinh mnh nht (cng l cc phn on c

mu cam m nht khi quan st bng mt thng).

Cn tbng kt qu 3.9 thyh dung mi chy ct c kh nng tch cc

khng sinh trn bn mng, nhng cha tch ring cc thnh phn trn ct

silicagel (do cc phn on thu c u cho 2 vt khng sinh trn sc k lp

mng). Do , cn phi kho st tm h dung mi c kh nng tch ring cc

thnh phn khng sinh.

Cng t bng 3.9 thy c t nht 3 khng sinh khc nhau trong hn hp

khng sinh th thu c. Chia cc phn on thu c thnh 2 phn: Phn I t

cc phn on 1-11 gm 2 khng sinh, y l phn chnh, c dng cho cc

nghin cu v sau, phn II t phn on 12-14 cng gm 2 khng sinh nhng

lng nhl cc phn on ph(mu rt nht).

Cc phn on phn I c mang gp chung, em ct quay chn khng

500C n khi bc hi ht dung mi, thu c 0,4859gkhng sinh tinh ch ln

1 (t y gi l khng sinh P1).

Nh vy, hiu sut tinh ch khng sinh ln th nht l:

H1% =

x 100% = 53,20 %

3.2.5. Kho st h dung mi tinh ch khng sinh ln th hai

T kt qu nghin cu trc [7], tc gi la chn h dung mi

ethylacetat: methanol (10:1) lm h dung mi chy ct tinh ch khng sinh.

Tuy nhin, khi kho st li chng ti thy kh nng tch ca h ny trn SKLM

cha tt,do chng ti tin hnh kho st tm ra h dung mi chy ct tthncho qu trnh tinh ch tip theo.

u tin, kho st mt s h dung mi bng cch thay i t l

ethylacetat. Cc h dung mi c kho st bao gm ethylacetat: methanol vi

cc t l l 20:1; 25:1; 30:1; 35:1. Kt qu SKLM cho thy h dung mi


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ethylacetat: methanol vi t l 20:1 cho kh nng tch cc khng sinh trn bn

mng tt nht, vt sc k trn, gn nht so vi cc h dung mi khc.

a h dung mi ethylacetat: methanol (20:1) ln ct Silicagel tch

hn hp khng sinh P1, thu 30 phn on, mi phn on 3ml, tin hnh SKLM

tng phn on thu c kt qu nh sau:

Bng 3.10: Kt qu SKLM cc phn on

Phn on S vt Rfvt 1 Rfvt 2

1 1 0,76 -

2-16 2 0,75 0,68

17-30 1 - 0,68

T bng 3.10 cho thy khi s dng h ethylacetat: methanol (20:1), cc vt

sc k tch tt trn bn mng nhng trn ct cc khng sinh tch vn cha tt.

Do cn ci tin h ethylacetat: methanol (20:1) tch cc khng sinh trn ct

tt hn.

Kho st cc nghin cu trc, thm mt dung mi na l n-hexan vo hethylacetat: methanol (20:1), tin hnh kho st tm t l n-hexan thch hp.

Bng 3.11. T l cc thnh phn h dung mi ethylacetat: methanol: n-

hexan

H dung miT l cc thnh phn

Etylacetat Methanol n-hexan

1.1 20 1 11.2 20 1 2

1.3 20 1 5


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Tin hnh sc k lp mng khng sinh P1 vi cc h dung mi 1.1, 1.2, 1.3 thu

c kt qu trong bng 3.12.

Bng 3.12. Kt qu SKLM vi cc h dung mi Ethylacetat: methanol: n-

hexan

H dung mi Rfvt 1 Rfvt 2

1.1 0,68 0,52

1.2 0,66 0,53

1.3 0,33 0,38

Kt qu bng 3.12 cho thy, h 1.1 so vi h ethylacetat: methanol

(20:1) ch c thm 1 lng rt nh n-hexan nhng ktqu tch ci thin nhiu,

cn h 1.2 c tc dng ko di khong cch gia 2 vt khng sinh nhng khng

khc h 1.1 nhiu thm ch cn km hn h 1.1. H 1.3 cha nhiu n-hexan nht

th dung mi khng ko c vt khng sinh P1 ln , cc vt khng sinh gn nh

khng tch nhau. Do , chng ti la chn h 1.1 tin hnh tinh ch khng

sinh P1.

3.2.6. Kt qu tinh ch khng sinh ln th hai

* Mc ch:

Tip tc tinh ch khng sinh P1 nhm thu c thnh phn khng sinh

tinh khit hn.

* Tin hnh v kt qu:

Khng sinh P1 c chy ct Silicagel bng h dung mi ethylacetat:

methanol: n-hexan vi t l 20:1:1 n khi dung mi chy rach cn mu vng

nht th thuc 30phn on mi phn on 3ml.

Tin hnh th hot tnh khng sinh tng phn on bng phng php

khoanh giy lc vi VSV kim nh l vi khun P. mirabilis. Kt qu ca th

nghim ny c th hin trn bng 3.13.


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Bng 3.13. Kt qu th HTKS cc phn on sau tinh ch ln 2.

Phn on D (mm) s Phn on D (mm) s

10,00 0,00

1621,76 0,44

211,13 0,82

1720,57 0,40

311,95 0,74

1818,86 0,55

416,37 0,48

1917,88 0,65

518,06 0,82

2016,77 0,30

618,83 0,56

2115,52 0,53

7

19,31 0,56

22

12,91 0,39820,53 0,52

2310,02 0,19

921,64 0,43

247,39 0,50

1022,53 0,38

255,97 1,53

1122,84 0,17

265,87 0,68

1223,25 0,89

270,00 0,00

1323,46 0,70

280,00 0,00

1422,24 0,89

290,00 0,00

1521,78 0,78

300,00 0,00

Cc phn on c hot tnh khng sinh c mang chy sc k lp mng vi h

dung mi 1.1. Kt qu nyc th hin trong bng 3.14.

Bng 3.14. Kt qu SKLM cc phn on sau tinh ch ln th hai

Phn on S vt Rfvt 1 Rfvt 22-7 1 vt 0.68 -

8-16 2 vt 0,68 0,53

17-26 1 vt - 0,53


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* Nhn xt

Bng 3.13 cho thy phn on u tin vbn phn on cui cng

khng c HTKS. Cc phn on cn li c HTKS kh cao, c bit l cc phn

on t 4 n 20.

Bng 3.14. cho thy cc phn on t 2 n 7 l cng 1 khng sinh c

Rf=0,68. Cc phn on t 17 n 26 cho cng 1 cht khng sinh c Rf=0,53.

Trong cc phn on t 8 n 16 cha2 khng sinh nhng cha tchring c.

Do , tin hnh gp cc phn on t 2 n 7, ct quay chn khng

nhit 500C n ht dung mi, kt tinh thu c 0,0423g khng sinh c

Rf=0,68 (t y gi l khng sinh X1).

Gp cc phn on t 17 n 26, ct quay chn khng nhit 500C

n ht dung mi, kt tinh thu c 0,0532g khng sinh c Rf=0,53 (t y gi

l khng sinh X2).

Th SKLM khng sinh X1 v X2 vi 4 h dung mi khc nhau th u

thy 1 vt trn, gn, khng ko ui. Nh vy, X1 v X2 l cht tinh khit cao.

Hiu sut tinh ch khng sinh X1 t khng sinh P1 l:

HX1=

x 100% = 8,71 %

Hiu sut tinh ch khng sinh X2 t khng sinh P1 l:

HX2=

x 100% = 10,95 %

Gp phn on t 8 n 16, ct quay chn khng nhit 500C n ht

dung mi, kt tinh thu c 0,2068g hn hpkhng sinh X1 v X2 ( gi l P2).

Nh vy, lng khng sinh trong cc phn on t 8 n 16l rt ln, cn tm

c dung mi tt hn tch khng sinh X1 v X2 t hn hp khng sinh P2

ny.

3.2.7. Kho st h dung mi tinh ch khng sinh ln th ba


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Tin hnh kho st cc h dung mi khc bng cch thay i thnh phn

methanol trong h 1.1 bng cc ancol mch di hn c th l ethanol v n-

propanol

Bng 3.15: Thnh phn cc h dung mitinh ch khng sinh ln 3

H dung mi Thnh phn T l

1.1 Ethylacetat: Methanol: n-hexan 20:1:1

2.1 Ethylacetat: Ethanol: n-hexan 20:1:1

2.2 Ethylacetat: n-Propanol: n-hexan 20:1:1

Tin hnh sc k lp mng khng sinh P2 vi cc h dung mi 1.1, 2.1, 2.2 thu

c kt qu trong bng 3.12.

Bng 3.16. Kt qu SKLM vi cc h dung mi tinh ch khng sinh ln 3

H dung mi Rfvt 1 Rfvt 2

1.1 0,68 0,55

2.1 0,58 0,40

2.2 0,35 0,31

* Nhn xt:Kt qu bng 3.16 cho thy, so vi h 1.1, th h 2.1 tch tt hn,

vt sc k trn, gn hn. Cn h 2.2 th cc cht gn nh l khng tch.Do ,

chng ti la chn h 2.1 tin hnh tinh ch khng sinh P2.

3.2.8. Kt qutinh ch khng sinh ln th ba

* Mc ch:

Tip tc tinh ch khng sinh P2 nhm thu c thnh phn khng sinh

tinh khit hn.

* Tin hnh v kt qu:


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Khng sinh P2 c chy ct Silicagel bng h dung mi ethylacetat:

ethanol : n-hexan vi t l 20:1:1 n khi dung mi chy rach cn mu vng

nht th thu 30phn on mi phn on 3ml.

Tin hnh th hot tnh khng sinh tng phn on bng phng php

khoanh giy lc vi VSV kim nh l vi khun P. mirabilis. Kt qu ca th

nghim ny c th hin trn bng 3.17.

Bng 3.17. Kt qu th HTKS cc phn on sau tinh ch ln 3

Phn on D (mm) s Phn on D (mm) s

10,00 0,00

1620,33 0,58

20,00 0,00

1720,01 1,31

30,00 0,00

1819,93 0,51

49,24 0,30

1918,86 0,55

511,25 0,67

2017,88 0,65

611,07 0,67

2115,84 0,55

716,03 0,80

2215,52 0,53

8 17,33 0,52 23 13,15 0,09

918,83 0,56

2410,02 0,19

1019,31 0,56

257,67 0,57

1120,85 0,44

266,31 1,53

1221,00 0,83

270,00 0,00

1321,88 0,58

280,00 0,00

14 21,99 1,33 29 0,00 0,00

1522,87 0,58

300,00 0,00

Cc phn on c hot tnh khng sinh c mang chy sc k lp mng

vi h dung mi 2.1. Kt qu ny c th hin trong bng 3.18.


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Bng 3.18. Kt qu SKLM cc phn on sau tinh ch ln th ba

Phn on S vt Rfvt 1 Rfvt 2

4-10 1 vt 0,58 -

11-15 2 vt 0,58 0,40

16-26 1 vt - 0,40

* Nhn xt

Bng 3.17 cho thy 3 phn on u tin v 4 phn on cui cng

khng c HTKS. Cc phn on cn li c HTKS kh cao, c bit l cc phn

on t 4 n 18.

Bng 3.18. cho thy cc phn on t 4 n 10 l cng 1 khng sinh c

Rf=0,58. Cc phn on t 16 n 26 cho cng 1 cht khng sinh c Rf=0,40.

Trong cc phn on t 11 n 15 cha 2 khng sinh.

Tin hnhnh ln tinh ch khng sinh th 2, gp cc phn on t 4 n

10, ct quay chn khng nhit 500C n ht dung mi, kt tinh thu c

0,0321g khng sinh c Rf=0,58 (k hiu N1). Gp cc phn on t 16 n 26,

ct quay chn khng nhit 500

C n ht dung mi, kt tinh thu c 0,0424g khng sinh c Rf=0,4 ( k hiu N2):

Hiu sut tinh ch khng sinh N1 t hn hp khng sinh P2 l:

HN1=

x 100% = 15,52 %

Hiu sut tinh ch khng sinh N2 t hn hp khng sinh P2 l:

HN2=

x 100% = 20,52 %

Th SKLM khng sinh N1, N2 ny vi cc khng sinh X1, X2 thu c

trn vicc h dung mi khc nhau thy cho Rftng t nhau, cc vt sc k

u trn, gn, do khng sinh N1 thu c chnh l khng sinh X1 ca ln tinh

ch th 2. V khng sinh N2 chnh l khng sinh X2 ca ln tinh ch 2. Gp


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khng sinh N1 vi khng sinh X1 bn trn thu c khng sinh tinh khit1 ( k

hiu l KS1).

Tng t nh vy, gp khng sinhN2 vi khng sinh X2 thu c khng

sinh tinh khit2 ( k hiu KS2).

Khi lngca khng sinh KS1sau ba ln tinh ch l:

mKS1= 0,0423 + 0,0321 = 0,0744 g

Hiu sut tinh ch khng sinh KS1 t khng sinh th l:

HKS1=

x 100% = 8,15 %

Khi lng ca khng sinh KS2 sau ba ln tinh ch l:

mKS2= 0,0532 + 0,0424 = 0,0956 g

Hiu sut tinh ch khng sinh 2 t khng sinh th l:

HKS2=

x 100% = 10,47 %

3.3. Nghin cutnh cht,cu trc khng sinh

3.3.1. Kt qu o nng chy

Th nghim o nng chy ca khng sinh KS1 v KS2 c thc hin

ti phng K thuttng hp Ha dc-b mn Cng nghip Dc,trng i

hc Dc H Ni. Kt qu nh sau:

Khng sinh KS1 nng chy 143-145 oC.

Khng sinh KS2 nng chy 231-236oC.

3.3.2. Kt qu ph t ngoi

Tin hnh o ph t ngoi ca khng sinh KS1 v KS2 ti khoa Ha l-

i hc Dc H Ni. Kt qu ph UV cho thy khng sinh KS1 c 3 nh hp

th bc sng 1 = 444 nm, 2 = 239,5 nm v 3= 203 nm.


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Khng sinh KS2 c 2 nh hp th bc sng 1 = 443,5 nm v bc

sng 2 = 204 nm.

Hnh nh ph UV ca KS1 v KS2 c th hin trong ph lc(Hnh P1, P2).

3.3.3. Kt qu ph hng ngoi

Ph IR ca 2 khng sinh KS1 v KS2 c o ti vin Ha hc bng

phng php vin nn KBr vng bc sng t 4000 500 cm-1.

Kt qu cho thy:

Ph IR ca cht KS1 c 12 bc sng hp th cc i l 3366,32 cm-1,

2931,99 cm-1

, 1731,56 cm-1

, 1655,90 cm-1

, 1577,44 cm-1

, 1462,55 cm-1

, 1266,39cm-1, 1101,06 cm-1, 980,57 cm-1, 733,98 cm-1, 588,26 cm-1v 479,13 cm-1.

Ph IR ca cht KS2 c 14 bc sng hp th cc i l 3393,74 cm-1,

3266,85 cm-1, 2963,72 cm-1, 1744,99 cm-1, 1644,30 cm-1, 1575,31 cm-1, 1478,20

cm-1, 1301,87 cm-1, 1197,10 cm-1, 1120,93 cm-1, 813,78 cm-1, 627,23 cm-1,

545,45 cm-1, 479,01 cm-1.

Bng 3.19: Bin gii cc nhm chc ca KS1 t ph IR

Bc sng

(cm-1)

Nhm chc c trng Bc sng

(cm-1)

Nhm chc c trng

3366,32 OH trong phenol, hoc

carboxylic acid.

-N-H trong amin hoc amid.

1266,39 -C-O trong alcol, ether,

ester, carboxylic, anhydrid.

-C-F.

S=O trong sulfon, sulfunyl

chlorid, sulfat, sufonamid.

2931,99 - C-H ca alkan. 1101,06 -C-O trong alcol, ether,

ester, carboxylic, anhydrid.

-C-F.


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S=O trong sulfoe, sulfunyl

chlorid, sulfate, sufonamid.

C-N ca amin

1731,56 -CO ca aldehyd, hoc ester. 980,57 -CH trong alken.

1655,90 -CO ca amid hoc C=N ca

imin hoc oxim.

C=C ca alken.

733,98 -C-Cl.

-C-H trong alken, hoc nhn

thm.

1577,44 - N-H trong amin hoc amid. 588,26 -C-Br hocC-I.

1462,55 - CH3trong alkan. 479,13 -C-Br hoc C-I.

Bng 3.20: Bin gii cc nhm chc ca KS2 t ph IR

Bc sng

(cm-1)

Nhm chc c trng Bc sng

(cm-1)

Nhm chc c trng

3393,74

3266,85

OH trong phenol, hoc

carboxylic acid.

-N-H trong amin hoc amid .

1301,87 -CN trong amin.

S=O trong sulfon, sulfunyl

chlorid, sulfat, sufonamid.

C-F.

2963,72 -C-H ca alkan. 1197,10

1120,93

-C-O trong alcol, ether, ester,

carboxylic, anhydrid.

-C-N ca amin.

S=O trong sulfon, sulfunylchlorid, sulfat, sufonamid

1744,99 -CO ca ester 813,78 -CH trong alken hoc nhn

thm.


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1644,30 -CO ca amide hoc C=N

ca imine hoc oxime

C=C ca alken

627,23 C-Cl

1575,31 -N-H trong amin hoc amid 545,45 C-X (X= Br, I)

1478,20 C=C trong nhn thm 479,01 C-X (X= Br, I)

* Nhn xt:Nh vy c th s b d on khng sinh KS1, KS2 c th c cc

nhm chc amin, amid, acid carboxylic, aldehyd, este, c nhn thm, cc mui

ca amin bc 2, bc 3....Hnh nh ph IR ca KS1 v KS2 c th hin trong

ph lc( Hnh P3, P4).

3.3.4. Kt qu o ph khi

Tin hnh o ph khi ca KS1 v KS2 ti Vin Ha hc. Kt qu cho

thy khi lng phn t d kin ca KS1 v KS2 ln lt l: 1269,45 vC v

1255,44 vC. Hnh nh ph MS ca hai cht ny c th hin trong ph lc

(Hnh P5, P6).

3.3.5. Kt qu o ph cng hng t ht nhn

Tin hnhh o ph cng hng t ht nhn i vi mu KS2, gm c: 1H

NMR, 13C NMR, DEPT, COSY, HSQC, HMBC.

Ph 13C-NMR xut hin 13 tn hiu ti vng trng thp t 160-180 ppm

ti 166,32; 166,485; 166,522; 166,601; 167,572; 167,671; 168,624; 169,046;173,256; 173,279; 173,317; 173,652; 179,063. Trong c 12 tn hiu u in

hnh cho s c mt ca 12 nhm C=O trong lin kt peptid N-C=O, v mt

nhm C=O trong vng ti 179,063


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Xut hin 12 tn hiu ca 12 nhm methyl ti 39,216; 39,144; 34,956;

34,889; 19,093; 19,070; 19,029; 18,985; 17,716; 17,313; 15,025; 7,767. Trong

, 4 tn hiu ti 39,216; 39,144; 34,956; 34,889 dch chuyn v trng thp

hn so vi tn hiu nhm methyl thng thng=> in hnh cho 4 nhm methyl

gn trc tip vi nguyn t Nit.

Cng t ph 13C-NMR , ph DEPT 135 xut hin tn hiu ca 7 nhm

CH2ti 22,838; 23,005; 30,981; 31,226; 47,350; 47,590; v 51,374. Trong

ngoi cc tn hiu 22,838; 23,005, cn li 5 tn hiu u dch chuyn mnh v

pha trng thp hn cc nhm methylene thng thng. iu ny chng t 5

nhm methylene ny u c lin kt vi cc nhm ht in t mnh nh Nit v

C=O.

Ph 13C-NMR , ph DEPT 90 xut hin tnhiu ca 16 nhm CH- ti

26,945; 27,008; 31,562; 31,804; 54,818; 55,171; 56,315; 56,554; 58,758; 58,918;

71,184; 71,384; 74,985; 75,053; 125,663; 130,276. Trong , ngoi 4 tn hiu ti

26,945; 27,008; 31,562; 31,804 th cc tn hiu khc u b dch chuyn mnh

v trng thp hn cc nhm CH- thng thng, chng t cc nhm ny nmtrong vng thm.

c bit ph 13C-NMR v 1H cho thy cc tn hiu ca khung

phenoxazone ti C=O: 179,063; CH3: 15,025 v 7,767; CH: 130,276, 125,663;

C: 147,496; 145,845; 145,067; 140,482; 132,606, 129,122; 127,660; 113,527;

101,800. Ph 1H cho hai tn hiu H: 7,607; 7,353, da vo tng tc trn ph

HSQC c th kt lun hai tn hiu l tn hiu ca 2 proton olefin ca khung

phenoxazone ti v tr octo ca khung (C7v C8).

Di y l bng so snh d kin ph NMR ca KS2 vi khung phenoxazone

ca phn t actinomycin D [16, 17, 20].


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Bng 3.21. So snh ph NMR ca KS2 vi khung phenoxazone ca

actinomycin D

V tr Actinomycin D KS2

1 101,6 101,800

2 146,0 145,845

3 179,1 179,063

4 113,5 113,527

4a 145,1 145,047

5a 140,5 140,482

6 127,5 127,660

7 130,2 130,276

8 125,9 125,663

9 132,6 132,606

9a 129,1 129,122

-CH3 v tr 4 7,7 7,767

-CH3 v tr 6 15,0 15,025

C=O v tr 3 179,1 179,063

H+ v tr 7 7,6 7,607

H+ v tr 8 7,3 7,353

Nh vy, c th kt lun, khng sinh KS2 phn lp t x khun

Streptomyces 173.113l khng sinh thuc nhm actinomycin.Kt qu o phNMR c th hin trong phn ph lc( T hnh P7 n

hnh P12).


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CHNG4. BN LUN


T nghin cu trc[7], sau khi tri qua bcsng lc ngu nhin v 3ln t bin bng UV v ha cht th hot tnh khng sinh ca chng

Streptomyces 173.113 tng r rt. La chn chng c hot tnh khng sinh cao

nht t nghin cu trc, tip tc tin hnh sng lc ngu nhin v t bin bng

UV tng hot tnh khng sinh.Mc d nghin cu trc, chng c hot tnh

khng sinh cao nht c ng knh vng v khun ln ti 28,19 mm(VSV kim

nh l S. aureus), v 26,12 mm (VSV kim nh l P. mirabilis), cn nghin

cu ny ng knh vng v khun ca chng c hot tnh khng sinh mnh

nht sau sng lc ngu nhin v 2 ln t bin ch t c 2 6,03 mm (VSV kim

nh l S. aureus), v 24,94 mm (VSV kim nh l P. mirabilis) nhng so vi

mu chng th vn cao hn ( iu ny c th do iu kin nhit , m lc

lc tin hnh nui cy khc nhau, nh hng ti ng knh vng v khun).

Qua ln t bin bng nh sng UV ln th nht, vi sng st l 0,73%

th a phn cc bin chng thu c c hot tnh cao hn so vi mu chng (t

bin dng). Trong , cao nht l bin chng B1.13 vi hot tnh khng sinh

bng 113,26%so vi mu chng.

Tip tct bin bin chng B1.13 bng nh sng UV ln th hai,

sng st l 0,58% th vn thu c t bin dng. Cao nht l bin chng

B2.02 c hot tnh khng sinh bng 110,26%so vi mu chng.

Nh vy, kt qu 2 ln t bin bng nh sng UV lm tng hot tnhkhng sinh ca chng x khun gc(hot tnh khng sinh sau 2 ln t bin bng

124,88% so vi mu ban u).

Do gii hn v thi gian, nn chng ti cha th tin hnh t bin bng

tc nhn khc nh gi s thay i hot tnh khng sinh ca x khun. V vy,


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chng ti s xut thay i tc nhn t bin cho nghin cu sau ny (c th

dng tc nhn ha hc).

4.2. Xc nh iu kin ln men, chit tch khng sinh ti thch

Tin hnh ci to ging qua sng lc ngu nhin v t bin UV, chng ti

la chn c 9 dng chng, bin chng tt la chn chng ln men tt

nht, l: SL.02, SL.09, SL.14, B1.04, B1.13, B1.30, B2.02, B2.10,

B2.25. Tin hnh ln men ring bit v so snh hot tnh khng sinh ca cc

dch lc dch ln men th thy bin chng B2.02ln men chm tt nht. Kt qu

ny l hp l bi v B2.02 l bin chng c HTKS cao nht thu c sau tbin ln hai.

Nng cao kh nng ln men ca chng / bin chng bng cch thm vo

mi trng MT1dt mt lng cao nm men. Kt qu thu c mi trng ln

men ti thch l MT1dt.2 (thm 0,5% cao nm men vo MT1dt), dch ln men

c hot tnh khng sinh cao hn dch ln men ca MT1dt ( hot tnh khng sinh

dch ln men MT1dt.2 bng 107,16% MT1dt).

Lng khng sinh trong dch ln men thu c l 0.1218 g/l, so snh vi

nghin cu trc lng khng sinh trong dch ln men ch t 0,1033 g/l [7].

Nh vy, qu trnh chn lc, ci to ging, thay i mi trng nui cy lm

tng hiu qu sinh tng hp khng sinh ca Streptomyces 173.113so vi nghin

cu trc.

Khng sinh th thu c sau ct quay l mt hn hp nhiu cht khng

sinh v cc tp cht khc (c th l cht mu,). Do , vic tch v tinh ch

khng sinh c ngha rt quan trng trong qu trnh nghin cu khng sinh.

Trong ti ny, khng sinh c tinh ch ba ln. Lnth nht, s dng

h dung mi butylacetat: aceton: triethylamin (1:2:1) tin hnh sc k ct.

H ny c tc dng gi li mt s tp cht (c th l cht mu trong khng sinh


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th, do trc chy ct khng sinh th c mu nu, sau chy ct c mu

ti). Tuy nhin, cc phn on khng sinh P1 sau khi sc k ct, mc d cc

khng sinh c tch nhau trn SKLM nhng li khng tch nhau trn sc k ct (

mi phn on u c t nht 2 cht khng sinh). Nh vy, cn phi tm h dung

mi khc c th tch ring c cc thnh phn khng sinh trn sc k ct.

Ln tinh ch th hai s dng h dung mi ethylacetat: methanol: n-

hexan ( 20:1:1) sc k ct tch cc thnh phn khc nhau ca hn hpP1.

Nhn vo bng kt qu 3.13 v 3.14 c th thy cc phn on t2 n 7,

t 17 n 26 cho ta c 2 khng sinh tinh khit l X1 v X2 c hot tnh khng

sinh kh cao. Tuy nhin, cc phn on xen ph gia cht X1 v X2 l cc phn

on 8 n 16 cng c hot tnh khng sinh rt cao (khi quan st bng mt

thng th cc phn on 2 cht ny c mu cam rt m), ct loi dung mi

thu c 0.2068g. Nh vy, phn on hn hp t 8 n 16 cn cha mt lng

lng khng sinh tinh khit rt ln m h ethylacetat: methanol: n-hexan (

20:1:1) cha tch c. Do , chng ti kho st tm ra h dung mi c

kh nng tch ring c khng sinh X1 v X2 tt hn.Thay i methanol trong h dung mi trn bng cc alcol c mch di hn

nhm tng thi gian lu trn ct ca cc cht, do d tch ring cc cht ra

hn. Kho st cc h, thu c h dung mi ethylacetat: ethanol: n-hexan (

20:1:1)c kh nng tch cc cht tt hn h s dng methanol. C th, hiu sut

ln chy ct th 3 ca cht khng sinh tinh khit 1 , v 2 ln lt l 15,52% v

20,52%, tng cao hn so vi hiu sut chy ct ln 2 ca cht 1, v 2 l 8,71%

v 10,95%. Cc khng sinh tch ra t ln tinh ch 3 cng chnh l cc khng sinh

tch ra t ln tinh ch 2. Gp ln lt cc khng sinh ca hai ln tinh ch thu

c hai khng sinh tinh khit KS1, v KS2


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Hiu sut tinh ch sau cng ca cc khng sinh KS1 v KS2 ln lt l

8,15% v 10,47%.So snh vi nghin cu trc, hiu sut tinh ch sau cng

ca khng sinh chnh l 33,48% tuy nhin, tinh khit ca khng sinh thu c

cha cao. Trong nghin cu ny, mc d hiu sut tinh ch cha bng nghin

cu trc, tuy nhin tinh khit cao hn do cc nghin cu v sau c

chnh xc cao hn, nht l trong vic xc nh cu trc khng sinh thu c.

Nh vy, sau 3 ln chy ct tinh ch thu c haicht tinh khit l KS1 v

KS2 v qu trnh nghin cu cng cho thy, chng c vai tr tng ng nhau

trong vic to thnh hot tnh khng sinh ca chng x khun ny, khng th

phn r u l chtchnh, u l cht ph.

4.3. Nghin cu cu trc khng sinh

* Kt qu o ph UV: cho thy KS1 c 3 nh hp th chnh tng ng

vi 3bc sng 1 = 444 nm, 2 = 239,5 nm v 3= 203 nm. KS2 c 2 nh hp

th chnh tng ng vi 2 bc sng maxl 1 = 443,5 nm v 2 = 204 nm.

Cc nh hp th ny tng ng vi s chuyn dch in t t n ln *.

Trong n l cp in t t do (khng lin kt) cc d nguyn t O, N, S,

halogen. Nh vy khng sinh nghin cu l hp cht c cha in t n v lin

kt . Hay ni cch khc trong cu trc khng sinh c th c nhn thm, ni i

lin hp, c cha d t, hoc kt hp cc yu t trn.

* Kt qu o ph IR:Ph IR ca cht KS1 v KS2 cho php d on cc

nhm chc c trng trong cu trc ha hc ca khng sinh KS1 v KS2.

Nh vy, t bng 3.19 v bng 3.20c th s b d on khng sinh KS1

v KS2 c th c cc nhm chc amin, amid, acid carboxylic, aldehyd, este, c

nhn thm, cc mui ca amin bc 2, bc 3....

Tuy nhin, nhn vo kt qu ph IR c th thy cu trc ha hc ca KS1

v KS2 ht sc phc tp vi nhiu nhm chc khc nhau, do cn tin hnh o


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thm cc ph khi, ph cng hng t ht nhn xc nh c chnh xc cu

trc ca KS1 v KS2

* Kt qu o ph NMR v MS:

T hnh nh cc ph NMR ca cht KS2 cho thy cu trc ca khng sinh

ny rt phc tp. Do hn ch v trnh v thi gian, trong nghin cu ny

chng ti ch bin gii c mt s nhm chc c trng ca KS2 m cha bin

gii c cu trc y ca KS2.

T qu trnh bin gii cho thy cu trc KS2 c mt ca khung

phenoxazone, mt khung c trng cho cu trc cc khng sinh thuc nhm

actinomycin.

Nh vy, c th kt lun, khng sinh KS2 phn lp t x khun

Streptomyces 173.113l khng sinh thuc nhm actinomycin.


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KT LUN V KIN NGH

Kt lunSau mt thi gian nghin cu, chng ti c bn hon thnh c mc

tiu ban u ca lun vn. Cc kt lun c th nh sau:

- Qua 2 ln t bin bng UV th hot tnh khng sinh ca Streptomyces

173.113 tng ln nhiu ( hot tnh khng sinh sau t bin bng 124,88% trc

t bin).

- Tng kh nng ln men bng cch cho thm cao nm men vo mi

trng ln men ban u vi t l 0,5%: kh nng ln men tng 107,16% so vi

MT1dt.

- Thay i h ethylacetat: methanol: n-hexan ( 20:1:1) ln tinh ch 2

bng h ethylacetat: ethanol: n-hexan ( 20:1:1) ln tinh ch 3 lm tng hiu

sut tinh ch khng sinh KS1 ( tng t 8,71% ln 15,52%) v KS2 (tng t

10,95% ln 20,52%).

- Tinh ch c 2 cht khng sinh c tinh khit cao l KS1 v KS2 vihiu sut ln lt l: 8,15% v 10,47%.

- Khng sinh KS1 c cc c im:

+ Tinh th hnh kim, mu cam.

+ Nng chy 143-145C.

+ Ph UV c 3nh hp th: 1 = 444 nm, 2 = 239,5 nm v 3=

203 nm.+ Ph IR cho thy cu trc ha hc rt phc tp vi rt nhiu nhm

chc

+ Khi lng phn t: 1269,45vC.

- Khng sinh KS2 c cc c im:


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+ Tinh th hnh kim, mu gch.

+ Nng chy 231-236C.

+ Ph UV c 2 nh hp th: 1 = 443,5 nm v 2 = 204 nm.

+ Ph IR cho thy cu trc ha hc rt phc tp vi rt nhiu nhm

chc.

+ Khi lng phn t: 1255,44vC.

+ tin hnh o ph NMR, kt lun thuc nhm khng sinh

actinomycin, tuy nhin cng thc cu to c th vn cha bin lun c

Kin ngh

T nhng kt qu thu c, chng ti xut tip tc pht trin ti

nghin cu su hn theo cc hng sau:

- Tip tc bin gii cu trc ca KS1v KS2.

- Tin hnht bin bng ha cht nng cao hn na hiu sut sinh

tng hp khng sinh.

-Nghin cu tm phng php tinh ch khng sinh cho hiu sut cao hn.


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TI LIU THAM KHO

Ting Vit

1.

BY T(2008), Vi sinh vt hc, Nh xut bn Gio Dc, H Ni, tr. 22-

87.

2.Nguyn Vn Cch (2004), Cng nghln men khng sinh, NXB Khoa hc

v kthut, tr 130-136.

3.

L Huy Chnh (2007), Vi sinh vt y hc,NXB Y hc, tr. 25-57

4.

Cc Qun l Dc (2010), Quy hoch chi tit pht trin cng nghip

Dc Vit

Nam giai on n nm 2020 v tm nhn n 2030, H Ni.

5.Nguyn Ln Dng (2001), Vi sinh vt hc,NXB Gio dc, tr. 39-67.

6. L Huy Dng (2010),Nghin cu ln men sinh tng hp khng sinh t

Streptomyces 168.27, Kha lun tt nghip dc s, Trng i hc Dc

H Ni, H Ni.

7.

L Th Thu Hin ( 2013), Gp phn nghin cu ln men sinh tng hp

khng

sinh nhStreptomyces 173.113.Kha lun tt nghip Dc skha 2008-

2013, Trng i hc Dc H Ni.

8.

L Gia Hy (2013), Cng nghsn xut khng sinh, NXB Khoa hc v k

thut, tr 161-193.9. TMinh Kong (2006),Kthut sn xut dc phm II, NXB Y hc, tp

2

10.

on ThNguyn (2009), Vi sinh vt,NXB Gio dc Vit Nam, tr. 7-37.

11.

Lng c Phm (1999), Cng nghvi sinh vt, NXB Nng nghip.

12.Nguyn Hunh Minh Quyn (2011), iu tra, nghin cu mt s hot

cht c


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khnng khng vi sinh vt v khng dng tbo ung th txkhun, i

hc Quc gia H Ni, tr. 16-46.

13.

Nguyn Vn Thch (2009), Cng nghsinh hc dc, NXB Gio dc

Vit Nam, tr. 35-57.

14.

Trn ThThanh (2001), Cng nghvi sinh, NXB Gio dc, tr. 40-52.

15.Cao Vn Thu (2008), Vi sinh vt hc, NXB Gio dc.

16.Cao Vn Thu, Phan Vn Kim, Bi Vit H (2010), Actinomycin X2,

cht

khng sinh c ln men tqu trnh sinh tng hp, Tp ch Ha hc, T.48

(4), tr 469-474.

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PHLC

Hnh P1: PhUV ca cht khng sinh KS1


68/92

Hnh P2: PhUV ca cht khng sinh KS2


69/92

Hnh P3: PhIR khng sinh KS1

Hnh P4: PhIR khng sinh KS2


70/92

Hnh P5: Phkhi MS khng sinh KS1

Hnh P6: Phkhi MS ca khng sinh KS2


71/92

HnhP7:Ph

HNMRcakhngsinh

KS2


72/92

HnhP7a:Ph

HNMRgincakhngsinhK

S2


73/92

Hn

hP7b:Ph

1HNMRgincakhngsin

hKS2


74/92

HnhP7

c:Ph

HNMRgincakhngsinhKS2


75/92

H n h

P 8 : P h

13 C N M R c a k h n g s i n h K S 2


76/92

HnhP8a:Ph

CNMRgincakhngsinhKS2


77/92

HnhP8b:Ph

13CNMRgincakhngsinhKS2


78/92

Hnh P9: PhCOSY ca khng sinh KS2


79/92

Hnh P9a: PhCOSY gin ca khng sinh KS2


80/92

Hnh P9b: PhCOSY gin ca khng sinh KS2


81/92

Hnh P9c: PhCOSY gin ca khng sinh KS2


82/92

Hnh P10: PhDEPT ca khng sinh KS2


83/92

Hnh P10a: PhDEPT gin ca khng sinh KS2


84/92

Hnh P10b: PhDEPT gin ca khng sinh KS2


85/92

Hnh P11: PhHSQC c

a khng sinh KS2


86/92

Hnh P11a: PhHSQC gin ca khng sinh KS2


87/92

Hnh P11b: PhHSQC gin ca khng sinh KS2


88/92

Hnh P12: PhHMBC ca khng sinh KS2


89/92

Hnh P12a: PhHMBC gin ca khng sinh KS2


90/92

Hnh P12b: PhHMBC gin ca khng sinh KS2


91/92

Hnh P12c: PhHMBC gin ca khng sinh KS2


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nghiên cứu nâng cao khả năng sinh tổng hợp kháng sinh từ streptomyces 173.113

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